Methods of fabricating polymeric structures incorporating microscale fluidic elements

ABSTRACT

The present invention generally provides improved methods of fabricating polymeric microfluidic devices that incorporate microscale fluidic structures, whereby the fabrication process does not substantially distort or deform such structures. The methods of the invention generally provide enhanced bonding processes for mating and bonding substrate layers to define the microscale channel networks therebetween.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of U.S. patent application Ser. No.09/590,661, filed Jun. 7, 2000, now U.S. Pat. No. 6,555,067 B1, which isa division of U.S. patent application Ser. No. 09/073,7 10, filed May 6,1998, now U.S. Pat. No. 6,123,798.

BACKGROUND OF THE INVENTION

As with the electronics and computer industries, trends in chemical andbiochemical analysis are moving toward faster, smaller and lessexpensive systems and methods for performing all types of chemical andbiochemical analyses.

The call for smaller systems and faster methods has been answered, inpart, through the development of microfluidic technologies, whichperform chemical and biochemical analyses and syntheses in extremelysmall-scale integrated fluid networks. For example, publishedInternational Patent Application No. WO 98/00231 describes microfluidicdevices, systems and methods for performing a large number of screeningassays within a single microfluidic device that is on the order ofseveral square centimeters. Such developments have been made possible bythe development of material transport systems that are capable oftransporting and accurately dispensing extremely small volumes of fluidor other materials. See Published International Application No. 96/04547to Ramsey.

By accurately controlling material transport among a number ofintegrated channels and chambers, one is able to perform a large numberof different analytical and/or synthetic operations within a singleintegrated device. Further, because these devices are of such smallscale, the amount of time for reactants to transport and/or mix, is verysmall. This results in a substantial increase in the throughput level ofthese microfluidic systems over the more conventional bench-top systems.

By reducing the size of these microfluidic systems, one not only gainsadvantages of speed, but also of cost. In particular, these smallintegrated devices are typically fabricated using readily availablemicrofabrication technologies available from the electronics industrieswhich are capable of producing large numbers of microfluidic devicesfrom less raw materials. Despite these cost savings, it wouldnonetheless be desirable to further reduce the costs required tomanufacture such microfluidic systems.

A number of reporters have described the manufacture of microfluidicdevices using polymeric substrates. See, e.g., Published InternationalPatent Application No. WO 98/00231 and U.S. Pat. No. 5,500,071. Intheory, microfabrication using polymer substrates is less expensive dueto the less expensive raw materials, and the ‘mass production’technologies available to polymer fabrication and the like.

However, despite these cost advantages, a number of problems exist withrespect to the fabrication of microfluidic devices from polymericmaterials. For example, because polymeric materials are generallyflexible, a trait that is accentuated under certain fabrication methods,e.g., thermal bonding, solvent bonding and the like, it is difficult toaccurately manufacture microscale structural elements in such polymericmaterials. In particular, the microscale structures are easily deformedunder manufacturing conditions, either due to applied pressures orrelaxation of the polymer matrix based upon its intrinsic structuralproperties.

Accordingly, it would generally be desirable to have a method offabricating microscale devices where the structural aspects of thedevice are not substantially perturbed during the fabrication process.The present invention meets these and other needs.

SUMMARY OF THE INVENTION

It is a general object of the present invention to provide methods offabricating polymeric microfluidic devices, and the devices fabricatedusing these methods. In a first aspect, the present invention providesfor methods of fabricating a microfluidic device comprising a firstsubstrate having a first planar surface, and a second substrate layerhaving a first planar surface, wherein the first planar surface of thefirst substrate comprises a plurality of microscale grooves disposedtherein. The first planar surface of the second substrate is heatedapproximately to the transition temperature of the first surface of thesecond substrate without heating the first surface of the firstsubstrate approximately to the transition temperature of the firstsurface of the first substrate. The first surface of the first substrateis then bonded to the first surface of the second substrate.

This invention also provides methods of fabricating a microfluidicdevice comprising a first substrate having a first planar surface, and asecond substrate layer having a first planar surface wherein the firstplanar surface of the first substrate comprises a plurality ofmicroscale grooves disposed therein, and the first planar surface of thesecond substrate has a lower transition temperature than the firstsurface of the first substrate. The first planar surface of the secondsubstrate is heated approximately to its transition temperature. Thefirst surface of the first substrate is then bonded to the first surfaceof the second substrate.

This invention also provides methods of fabricating microfluidic devicescomprising a first substrate having a first planar surface, and a secondsubstrate layer having a first planar surface, wherein the first planarsurface of the second substrate has a lower transition temperature thanthe first surface of the first substrate. The first surface of thesecond substrate is heated approximately to the transition temperature.The first surface of the first substrate is bonded to the first surfaceof the second substrate.

This invention also provides methods of fabricating a microfluidicdevice comprising a first substrate having at least a first surface anda second substrate having at least a first surface, wherein at least oneof the first surface of the first substrate or the first surface of thesecond substrate comprises a textured surface, and mating and bondingthe first surface of the first substrate to the first surface of thesecond substrate.

This invention also provides methods of fabricating a microfluidicdevice comprising a first substrate having a first planar surface, and asecond substrate layer having a first planar surface, wherein the firstplanar surface of the second substrate has a lower transitiontemperature than the first surface of the first substrate. The firstsurface of the first substrate is thermally bonded to the first surfaceof the second substrate, whereby the first surface of the secondsubstrate does not substantially project into the plurality of channels.

This invention also provides a microfluidic device comprising a firstpolymeric substrate having at least a first planar surface, the firstplanar surface comprising a plurality of channels disposed therein. Thedevice also includes a second polymeric substrate layer having at leasta first planar surface, the first planar surface of the second substrateis bonded to the first planar surface of the first substrate, andwherein the first surface of the second substrate has a lower transitiontemperature than the first surface of the first substrate.

This invention also provides a microfluidic device comprising a firstpolymeric substrate comprising a first planar surface having a pluralityof microscale channels disposed therein. The device also contains asecond polymeric substrate comprising a first planar surface, the firstplanar surface of the second substrate being non-solvent bonded to thefirst planar surface of the first substrate, wherein the first surfaceof the second substrate does not substantially project into theplurality of channels.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic illustration of a microfluidic deviceincorporating a layered structure.

FIG. 2 illustrates examples of channel deformation in some methods offabricating layered polymeric microstructures. FIG. 2A illustrates theextrusion of a cover layer substrate into a channel structure fabricatedinto the surface of another substrate when the two substrates arethermally bonded together using conventional means. FIG. 2B illustratesthe softening or dulling of channel corners in a thermally bondedpolymeric microfluidic device, where the channel bearing structure isinjection molded, or otherwise has residual stresses frozen into thestructure.

FIG. 3 illustrates an example of surface texturing utilized to fabricatelayered polymeric microstructures. FIGS. 3A and 3B illustrate thebonding layer both before and after the bonding process, respectively.

FIG. 4 illustrates a plot of both structural deformation of surfacetextures in the mating of two substrates as well as local pressure onthe raised portions of the textures over time of the thermal bondingprocess.

FIG. 5 is a cross-section of two channels thermally bonded together.FIG. 5A illustrates a channel in which an upper substrate is protrudinginto the channel, whereas the channel shown in FIG. 5B is substantiallyclear of obstruction from the upper substrate.

DETAILED DESCRIPTION OF THE INVENTION

I. General

As noted above, the present invention generally provides improvedmethods of fabricating polymeric microfluidic devices. Generally, theseimproved methods allow for the rapid fabrication of polymeric devicesthat incorporate microscale fluidic structures, whereby the fabricationprocess does not substantially distort or deform such structures.

As used herein, the term “microscale” or “microfabricated” generallyrefers to structural elements or features of a device which have atleast one fabricated dimension in the range of from about 0.1 μm toabout 500 μm. Thus, a device referred to as being microfabricated ormicroscale will include at least one structural element or featurehaving such a dimension. When used to describe a fluidic element, suchas a passage, chamber or conduit, the terms “microscale,”“microfabricated” or “microfluidic” generally refer to one or more fluidpassages, chambers or conduits which have at least one internalcross-sectional dimension, e.g., depth, width, length, diameter, etc.,that is less than 500 μm, and typically between about 0.1 μm and about500 μm. In the devices of the present invention, the microscale channelsor chambers preferably have at least one cross-sectional dimensionbetween about 0.1 μm and 200 μm, more preferably between about 0.1 μmand 100 μm, and often between about 0.1 μm and 20 μm. Accordingly, themicrofluidic devices or systems prepared in accordance with the presentinvention typically include at least one microscale channel, usually atleast two intersecting microscale channels, and often, three or moreintersecting channels disposed within a single body structure. Channelintersections may exist in a number of formats, including crossintersections, “T” intersections, or any number of other structureswhereby two channels are in fluid communication.

In particularly preferred aspects, the microfluidic devices describedherein, are used in conjunction with controlled electrokinetic materialtransport systems, as described in Published International ApplicationNo. 96/04547 to Ramsey, which is incorporated herein by reference forall purposes. Specifically, such material transport systems are used totransport fluid and/or other materials through the interconnectedchannels of the devices in a controlled fashion.

The microfluidic devices in accordance with the present inventioninclude a body structure that has disposed therein, an integratednetwork of microscale channels or conduits. The different elements ofthe body structure may be fabricated from a number of different separateparts to define the various channels and/or chambers of the device. Inparticularly preferred aspects, the body structure of the device isfabricated as a layered structure. An example of a device incorporatingthis layered structure is illustrated in FIG. 1. In particular, thedevice 10, includes a bottom portion 12 which comprises a solidsubstrate that is substantially planar in structure, and which has atleast one substantially flat upper surface 14.

The channels and/or chambers of the microfluidic device are typicallyfabricated into the upper surface of the bottom substrate or portion 12,as microscale grooves or indentations 16, using the microfabricationtechniques described herein. The top portion or substrate 18 alsocomprises a first planar surface 20, and a second surface 22 oppositethe first planar surface 20. In the microfluidic device shown in FIG. 1,the top portion of the device optionally includes a plurality ofapertures, holes or ports 24 disposed therethrough, e.g., from the firstplanar surface 20 to the second surface 22 opposite the first planarsurface.

The first planar surface 20 of the top substrate 18 is then mated, e.g.,placed into contact with, and bonded to the planar surface 14 of thebottom substrate 12, covering and sealing the grooves and/orindentations 16 in the surface of the bottom substrate, to form thechannels and/or chambers (i.e., the interior portion) of the device atthe interface of these two components. In those embodiments utilizingincorporated reservoirs or ports, the holes 24 in the top portion of thedevice are oriented such that they are in communication with at leastone of the channels and/or chambers formed in the interior portion ofthe device from the grooves or indentations in the bottom substrate. Inthe completed device, these holes function as the reservoirs forfacilitating fluid or material introduction into the channels orchambers of the interior portion of the device, as well as providingports at which electrodes may be placed into contact with fluids withinthe device, allowing application of electric fields along the channelsof the device to control and direct fluid transport within the device.

As noted above, at least one, and preferably both or all of thesubstrate layers, e.g., as described with reference to FIG. 1, comprisea polymeric material or substrate. In accordance with the presentinvention, the polymeric substrate materials used to fabricate themicrofluidic devices described herein are typically selected from a widevariety of different polymeric materials. Examples of particularlyuseful polymer materials include, e.g., polymethylmethacrylate,polycarbonate, polytetrafluoroethylene, polyvinylchloride,polydimethylsiloxane, polysulfone, polystyrene, polymethylpentene,polypropylene, polyethylene, polyvinylidine fluoride, andacrylonitrile-butadiene-styrene copolymer.

Because microscale fluidic structures are of such small dimensions,e.g., channel depths typically falling in the range of from about 1 to50 μm, even slight deformation of a channel's structure can haveseriously adverse effects on the function of the device incorporatingthat channel, including partial or total channel occlusion, formation ofsharp corners in the channels along which irregular capillary flowoccurs, structural irregularities causing disruptive flow patternsduring operation, and the like.

Unfortunately, channel distortion of the type referred to above, isexactly the type of problems faced in fabricating polymeric microfluidicdevices. In particular, in preferred aspects, the microfluidic devicesof the present invention are fabricated as an aggregation of differentsubstrate layers that are typically planar in structure. One of thelayers typically includes a series of grooves and/or depressionsfabricated into its surface, which grooves or depressions define thechannels and chambers of the ultimate microfluidic device. A secondlayer is overlaid and bonded to the first layer to seal the grooves anddepressions forming the channels and chambers. Optionally, the channelsand/or chambers are defined in an intermediate layer, which defines thesides of the channels and/or chambers. The intermediate layer is thensandwiched and bonded between the top and bottom layers, which form thetop and bottom surfaces, respectively, of the channels and/or chambers.The substrate layers are then bonded together using known bondingtechniques. For polymeric substrates, such techniques include, e.g.,thermal bonding, ultrasonic bonding or welding, adhesive bonding, orsolvent bonding.

In thermal bonding of solid polymeric substrates, one or more of thesubstrates to be bonded is heated to the transition temperature of thesubstrate surface. As used herein, the “transition temperature” refersto the temperature at which the polymer substrate material, whichnormally has a glass-like character, undergoes the transformation from arigid material to a soft rubber, e.g., the melting point. In particular,as a polymer is heated to a temperature at or just below the transitiontemperature, the polymer starts to soften. In the case ofnon-crystalline polymeric materials, the transition temperature istypically referred to as the “glass transition temperature,” typicallydenoted by T_(g). At the glass transition temperature, the glass-likepolymer begins to take on the more rubbery character.

For non-polymeric substrates, e.g., glass, quartz, silicon and the like,the substrate is typically sufficiently hard that even under extremelyhigh bonding temperatures, e.g., in excess of 500° C., there issubstantially no deformation of the microscale channels between thesubstrates being bonded. For polymeric substrates, however, substantialdeformation can occur during thermal bonding at substantially lowertemperatures.

For example, when polymeric substrates are heated to their transitiontemperature and bonded together, microscale structural elements have atendency to flatten under the elevated temperatures and pressures.Similarly, otherwise flat substrate layers have a tendency to beextruded into cavities, depressions or grooves on the opposite substratesurface, e.g., channels and/or chambers, as a result of their softercharacter and the effects of the applied pressure. This extrusion of anupper substrate layer into a channel or chamber creates a number ofproblems. For example, such extrusion results in unknown or variablevolumes for the channels and chambers, and also results in substantialocclusion of channels. Further, and as referenced above, this channelextrusion can result in the generation of fluid shooters, where fluidsin the corners of channels move much faster than the remainder of thefluid. These shooters have a tendency to travel far ahead of the bulkfluid front in capillary filling of channels, and join together to trapair bubbles within the channels. The presence of such air bubbles,particularly in extremely small-scale channels can be fatal to theproper operation of the device.

In the case of injection molded polymeric parts, additional problems areassociated with the fabrication of polymeric devices. For example, inthe injection molding process, polymeric material injected into a moldhas a tendency to align the individual polymer molecule strands in themolded product in the direction of polymer injection. This alignment ofpolymer molecules results in an inherent or “frozen” stress in thehardened product as the polymer strands tend toward their natural randomstate. This frozen stress often results in a disproportionate shrinkingof the molded part in the length dimension of the aligned polymers, ascompared to the width, when the parts are heated to or near theirtransition temperatures, e.g., for thermal bonding. This shrinking thenleads to deformation of microscale structures on the polymer part, andeven warping of the part as a whole.

FIG. 2 illustrates some examples of the types of channel deformationthat occur during these types of thermal bonding processes for polymericsubstrates. FIG. 2A illustrates the extrusion of an upper substratelayer into a channel structure fabricated on the lower substrate layerfollowing thermal bonding of the substrates. Although illustrated as adrawing, the dimensions provided represent actual and substantialencroachment of the upper substrate into the channel. This was a resultof heating the layers to above the transition temperature for thematerial used, and applying pressure to the two substrates to facilitatebonding. As shown, the upper layer encroaches upon the channel structureresulting in reduced performance of the device incorporating thischannel, as described above.

FIG. 2B illustrates an example of thermally bonded polymeric substrateswhere the lower substrate, bearing the channel structure was injectionmolded, or otherwise had stresses frozen into it. Relaxation of thepolymers in the substrate when the substrate was heated during thermalbonding resulted in a dulling of the channel edges.

One alternative to thermal bonding is ultrasonic welding or bonding. Inthese methods, a series of sharp protrusions or ridges (“energydirectors”) are fabricated on one of the parts to be bonded. Underelevated pressure and high frequency vibrations, these energy directorsmelt and bond with the corresponding surface on the other substrate.Again, however, use of such methods generally results in excessivechannel distortion or irregularity, such that such the methods are notuseful in fabrication of microscale fluidic devices. In particular, theedges of the bonded regions resulting from these ultrasonic methods tendto be relatively irregular in comparison with the edges of the channels.As such, the corners at which the two substrates meet will also beirregular, as a result of some material encroaching into the channel,and/or openings where the bonded edge does not reach the channel edge.These latter irregularities cause substantial difficulty in microfluidicsystems as they can give rise to fluid “shooters” (edges of a channel atwhich capillary flow is faster than capillary flow in the rest of thechannel) during fluid introduction and movement within the channel.

Another alternative to thermal bonding is the use of adhesives to bondpolymeric parts together. The use of adhesives alleviates the problemsof thermal deformation of channel structures. However, in order to beeffective in the fabrication of microfluidic systems, adhesive must becarefully applied in order to ensure that the channels and chambers willbe entirely sealed after bonding. Further, because microfluidic devicesare generally used in sensitive analytical operations, it is generallydesirable to avoid introducing any unwanted chemical components into thechannels and/or chambers of the device. Thus, while one must ensureapplication of adequate adhesive to ensure sealing, one must avoidgetting the adhesive into the extremely small scale channels andchambers. In addition to adverse chemical interactions, suchcontaminants can potentially produce structural barriers or occlusionswhich adversely affect fluid movement.

Another method of bonding polymeric substrates is through the use ofsolvent bonding processes. Typically, these processes involve the matingof two polymeric parts followed by application of a polymer softeningsolvent to the space between the parts, e.g., via capillary action. Thesoftening and re-hardening of the polymer interface results in a bondedpart. Solvent bonding methods are well known in the art and aredescribed in, e.g., Plastics Technology, Robert V. Milby (McGraw-Hill1973), and Handbook of Plastics Joining: A Practical Guide (PlasticsDesign Library, 1996), both of which are incorporated herein byreference. The same contamination problems associated with adhesivebonding are also present in solvent bonding methods. Further, suchsolvent process typically cause at least some level of polymer softeningwhich can lead to adverse structural effects, e.g., as described above.In addition, solvent bonding processes will often produce stresscracking when used in conjunction with injection molding processes.

II. Polymer Selection

In a first aspect, the methods of the present invention generallyaddress the problems typically associated with the fabrication ofmicrofluidic devices from polymeric substrates. In preferred aspects,the methods described herein are directed to thermal bonding methods offabricating microfluidic devices. Accordingly, the methods of theinvention are generally described with reference to the fabrication ofmicrofluidic devices that incorporate a layered structure. Such devicestypically include a top portion, a bottom portion, and an interiorportion that is defined by the mating of the top portion to the bottomportion. Typically, a first substrate is provided which includes atleast one planar surface. The microscale structural elements of thedevice are generally fabricated into the first surface of the firstsubstrate. In the case of microscale fluidic channels and/or chambers,the structures typically are fabricated as microscale grooves ordepressions in that surface.

In addition to the channel structures of the device fabricated into thefirst substrate surface, the second substrate also typically includes aplurality of apertures disposed through it. Each aperture is generallyprovided so as to be placed in fluid communication with at least onechannel that is disposed within the interior portion of the device whenthe layers are bonded together. These apertures then function as thefluid reservoirs of the device, as well as points of access to thechannel structures, e.g., for fluid introduction, electrical sensing andcontrolled electrokinetic material transport, and the like.

Fabrication of the grooves in the substrate surface is generally carriedout using known polymer fabrication methods, e.g., injection molding,embossing, or the like. In particular, master molds or stamps areoptionally created from solid substrates, such as glass, silicon, nickelelectroforms, and the like, using well known microfabricationtechniques. These techniques include photolithography followed by wetchemical etching, LIGA methods, laser ablation, thin film depositiontechnologies, chemical vapor deposition, and the like. These masters arethen used to injection mold, cast or emboss the channel structures inthe planar surface of the first substrate surface. In particularlypreferred aspects, the channel or chamber structures are embossed in theplanar surface of the first substrate.

By embossing the channel structures into the first substrate, one avoidsthe stress relaxation problems associated with injection moldedsubstrates. In particular, because embossed substrates are not flowed orinjected into a mold, there is substantially less alignment of thepolymer strands from flowing of the polymer material. Accordingly,during thermal bonding, there is substantially less relaxation of theoverall substrate when the substrates are mated, and therefore,substantially less channel deformation.

Typically, the grooves fabricated into the surface of the firstsubstrate are fabricated as a series of intersecting grooves, to formthe integrated intersecting channel structures of the devices of theinvention. The grooves are formed into channels by mating a secondsubstrate layer to the first, to cover and seal the grooves and/ordepressions to form the channels and/or chambers of the device. Inaccordance with one aspect of the invention, the second substrate isthermally bonded to the surface of the first substrate over thechannels. The surfaces of the two substrates are typically planar topermit adequate contact across the surface.

In order to avoid additional distortion of channel structures on thefirst substrate during the thermal bonding of the second substrate, thefirst and second substrates are typically selected to have differingtransition temperatures. In particular, the substrate that bears themicroscale structures is typically selected to have a higher transitiontemperature than the cover layer that is to be bonded to it. Selectionof the channel bearing substrate to have a higher transitiontemperature, allows the cover layer to be heated to its transitiontemperature and mated with the channel bearing substrate, withoutdistorting or deforming the channel structures on the channel bearingsubstrate. Of course, depending upon the desired goal, the channelbearing substrate may be selected to have a lower transitiontemperature, e.g., if substrate extrusion into the channels is the mostcritical, or only actual problem to be addressed. In particularlypreferred aspects, both substrate layers are selected to minimize bothchannel distortion and channel occlusion problems, by selectingsubstrates that are sufficiently different in their transitiontemperatures to prevent channel distortion, but sufficiently close toprevent excessive extrusion of the upper substrate into the channelstructures. Selection of a polymer having a higher transitiontemperature for the channel bearing substrate, permits the use ofinjection molded parts. Specifically, because these substrates do notneed to be heated to their transition temperatures for thermal bonding,there is less chance of the substrate relaxing, and thus, resulting indeformation of the channels.

In preferred aspects, the transition temperature of the two substratesare at least about 5° C. apart, more preferably at least about 10° C.apart, more preferably, at least 20° C. apart, often at least 50° C.,and in some cases, at least 100° C. apart. For example, where onesubstrate (that having the lower transition temperature) has atransition temperature of approximately 80° C., the other substrate willtypically have a transition temperature of at least 85° C., preferablyat least 90° C., more preferably at least 100° C., often at least 130°,and in some cases at least 180° C. Generally speaking, the transitiontemperature of the substrate having the higher transition temperature istypically at least 40° C., while the transition temperature of thesubstrate having the lower transition temperature is less than 150° C.Alternatively, the surface of one substrate is heated to its transitiontemperature while the surface of the other substrate is maintained at alower temperature. As above, the first substrate is typically heated toa temperature at least 5° C., 10° C., 20° C., 50° C. or even at least100° above the temperature at which the other substrate is maintained.

Thus, in accordance with the methods described herein, the planarsurface of one of the substrates, typically the cover layer substrate,is heated approximately to the surface's transition temperature, withoutreaching the transition temperature of the surface of the othersubstrate, e.g., the channel bearing substrate. Typically, the entirepolymeric part is fabricated from a single polymer, and thus thetransition temperature of the surface is the same as the remainder ofthe substrate. However, it will be appreciated that multilayer polymericmaterials are also envisioned in accordance with the present invention,including polymer substrates bearing a different polymer coating.

Following the heating of the substrates to the first transitiontemperature, the substrates are bonded together. In most but not allcases, this typically involves the application of slight pressure to thetwo substrates, pressing their bonding surfaces together, to ensureadequate and complete bonding of the parts. In those cases where apressure is applied between the substrates, the amount of appliedpressure is typically dependent upon the polymers and temperatures used.However, in general, the applied pressures are generally in the range offrom about 0.1 kg/cm² to about 20 kg/cm².

In preferred aspects, the polymeric substrate materials used inaccordance with this aspect of the invention comprisepolymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene(TEFLON™), polyvinylchloride (PVC), polydimethylsiloxane (PDMS),polysulfone, polystyrene, polymethylpentene, polypropylene,polyethylene, polyvinylidine fluoride, ABS(acrylonitrile-butadiene-styrene copolymer), and the like. In manyaspects, the present invention utilizes those polymers which aregenerally non-crystalline in structure, i.e., polymethylmethacrylate,polycarbonate, polyvinylchloride, polydimethylsiloxane, polysulfone,polystyrene, polymethylpentene, polyvinylidine fluoride, andacrylonitrile-butadiene-styrene copolymer

In particularly preferred aspects, both substrates comprisepolymethylmethacrylate grades. In order to provide a differenttransition temperature for the second substrate, or cover layer, thissubstrate typically comprises an injection moldable grade of PMMA.Examples of such polymeric materials include, e.g., Acrylite polymers,e.g., M-30, L-40, etc., available from CYRO Industries, for injectionmoldable grades of PMMA and Plexiglas V-825, available from Atohaas,North America, for the structure bearing substrate, which has a highertransition temperature. Typically, adjustment of the transitiontemperature is accomplished through the adjustment of the polymercomposition, i.e., incorporating different amounts of other comonomers.For example, for PMMA, lower transition temperatures, e.g., forinjection moldable grades of PMMA, are generally achieved byincorporating other acrylic comonomers, i.e., ethylacrylate,methylacrylate or butylacrylate monomers, during the synthesis of thepolymer. Similarly, for polycarbonate polymers, transition temperaturesare generally adjusted by incorporation of bisphenol analogs duringsynthesis, and adjusting their relative concentration. In the case ofABS polymers, transition temperatures may be adjusted by adjusting therelative level of the polymers in the combination, e.g., acrylonitrile,butadiene and styrene. Optionally, with the addition of other additives,i.e., tackifiers, waxes and the like, one can increase adhesiveproperties of substrate surfaces at or below the transition temperatureof the bulk substrate material, thereby giving the surface of thesubstrate a lower effective transition temperature or “bondingtemperature.”

Transition temperatures are then adjusted by adjusting the relativepercentages of these other monomers, i.e., increasing to reducetransition temperature. Typically, these additional monomers are presentin the overall polymer at a concentration of at least about 0.1%, 1%, 2%and often at least about 5% or 10% or even greater, based upon the totalmonomer concentration used in the polymer synthesis, depending upon thedesired transition temperature range.

Alternatively, or additionally, transition temperatures for polymers maybe adjusted by adjusting the molecular weight of the polymers. Inparticular, longer and larger polymers typically have higher transitiontemperatures than smaller, shorter polymers. Thus, a substratefabricated from a polymer having a lower average molecular weight, has alower transition temperature than a polymer having a higher averagemolecular weight. In such cases, the polymer having the larger averagemolecular weight (and higher transition temperature) is at least about5% larger than the average molecular weight of the other substrate(having the lower transition temperature), preferably, at least about10% larger, more preferably at least about 20% larger, 50% and often atleast 100%, and in many cases, at least about 200% larger than thepolymer used to fabricate the other substrate having the lowertransition temperature.

As noted above, the methods of the present invention result in thefabrication of microfluidic devices where the channel structures are notsubstantially distorted. In addition, these devices are characterized inthat the cover layer substrate that is bonded to the channel bearingsubstrate does not substantially encroach upon, occlude or otherwiseproject into the channels of the device. The phrase “does notsubstantially project into the channel,” as used herein, means that thecross-sectional area of the channel structure as it is defined by thestructure bearing substrate (width of fabricated channel X depth offabricated channel), is not substantially blocked by extrusion of thecover layer substrate into the channel. Such occlusion is shown in FIG.2A. Typically, the cover layer occludes the cross-sectional area of thechannels by less than 20% of the potential channel cross-section. Inpreferred aspects, the occlusion is less than 10% of the totalcross-sectional area of the channel, and more preferably, less than 5%of the total cross-sectional area, and still more preferably, less than2% occlusion of the total cross-sectional area. While solvent bondingmethods are generally capable of producing devices where the cover layerdoes not substantially occlude the channels of the device, such solventbonding methods have a number of other disadvantages, as describedabove. In the present invention, such non-occluded channels arefabricated in non-solvent bonding and/or non-adhesive bonding methods,e.g., bonding methods that do not utilize solvents or adhesives, i.e.,thermal bonding, ultrasonic bonding, or the like.

III. Surface Textures

In an alternate aspect, the present invention provides methods offabricating microfluidic devices from polymeric substrates by providingat least one of the substrates with a textured surface to assistbonding. In particular, as noted above, the use of excessivetemperatures and/or excessive pressures during thermal bonding ofpolymeric substrates often results in deformation of the channelstructures, and/or occlusion of the channels by the upper substratelayers being extruded into the channels. Like the above describedaspects, the present embodiment of the invention improves thermalbonding and other methods of bonding polymeric substrates by reducingthe temperatures and pressures to which a substrate is exposed duringthe bonding process. In accordance with this aspect of the invention,pressures and/or temperatures for bonding are minimized by reducing theeffective surface area at which bonding occurs. In particular, themethods of the present invention provide one or both of the substratelayers having a textured bonding surface. By “textured bonding surface”is meant that the surface of the substrate that mates with and is bondedto the other substrate includes a structural texturing, such as a seriesof raised ridges, pillars, posts, or the like, that are disposed on thesurface in question. Texturing of the bonding surfaces may take on avariety of forms. For example, the texturing optionally includes aseries of parallel raised ridges/grooves fabricated into the bondingsurface. Other textures are also useful in accordance with the presentinvention, including raised ridges fabricated in a grid or diamondpattern, raised pillars or posts fabricated in sufficiently closeproximity that upon bonding, the spaces between them will be filled inand sealed.

In particularly preferred aspects, the surface texture is applied to thebonding surface of the substrate bearing the channel structures.Specifically, the microfabrication steps applied to the manufacture ofthe channel structures, i.e., embossing, injection molding, etc., can beexploited in the fabrication of the surface texturing. In addition, inpreferred aspects, the surface texture is applied to the surface intowhich the channel structures are fabricated. As such, the texture is notpresent within the channel itself, e.g., as would be the case if thetexturing was applied to the cover layer substrate. The texturing may beapplied uniformly over the entire bonding surface of interest.Alternatively, the texturing may be applied only in those areas wheresealing is desired, e.g. immediately surrounding the channels andchambers of the device.

Because the channel structures that are defined within the devices ofthe present invention have depths that typically range from about 5 μmto about 100 μm, it is generally desirable to provide surface texturinghaving substantially less depth. In preferred aspects, the texturing isprovided having a height (or depth) that is from about 1% to about 50%of the channel depth, and preferably, from about 1% to about 30% of thechannel depth, and still more preferably, between about 1% and about 10%of the channel depth. Accordingly, while the texturing may varydepending upon the depth of the channels of the device, the surfacetexturing as described herein will typically range from about 0.1 μm toabout 50 μm high (or deep), and preferably, from about 0.25 μm to about30 μm, and more preferably, from about 0.25 μm to about 10 μm high (ordeep). For channels that are on the order of 10 to 20 μm deep, surfacetexturing of between about 0.5 to about 2 μm in depth is generallypreferred.

In thermal bonding methods, the surface texturing serves to providelocalized areas at which melting and bonding occur between substratelayers, preventing such occurrences within the channel structures perse, and thus preventing substantial channel distortion. In particular,because pressure between two substrates is concentrated in the raisedtexture structures it requires a lower overall substrate temperature toproduce the desired bonding between the substrates, e.g., the combinedpressure and temperature effects are concentrated at the raisedridges/structures. Further, as the texture structures are melted andflattened during the bonding process, the amount of surface area incontact between the two substrates increases, thereby reducing thelocalized pressure/heating effects. This increase in surface area andeffective decrease in the localized pressure creates a bonding processthat is somewhat self-regulating. In particular, after the surfacetexturing is distorted or flattened enough by the heat and pressure, thecontact area between the substrates increases, thereby effectivelyreducing the localized pressure, which results in a considerable slowingof deformation. Specifically, the constant force applied to the texturestructures is dissipated over a larger substrate surface as thesetextures collapse into the rest of the substrate surface, therebyarresting the melting and bonding process.

This self-regulating process is illustrated in FIG. 4, which is asuperimposed graph of localized pressure versus time 402 and texturedeformation versus time 404. In particular, the local pressure at theinterface of two substrates, e.g., at the top of the texturing (ridges,posts, etc.) at the beginning of the thermal bonding process, is spreadover only the area of the interface. As the texturing (ridges, posts,etc.) melts during the thermal process, the area of the interfaceincreases as the texturing flattens out. Accordingly, the same amount ofapplied force is spread over a wider area, until the texturing is nearlycompletely flattened out, at which point the pressure at the interfacingsurfaces stabilizes at nor near the total applied pressure (as theinterface is substantially a single surface, thus local pressure=totalpressure).

FIG. 3A illustrates the use of surface texturing in the bonding methodsdescribed herein. As shown, the upper substrate/cover layer 302 is matedwith the lower substrate 304 that includes a channel 306 fabricated intoits surface. The upper surface 308 of the bottom substrate 304 hasprovided thereon a surface texturing that includes a plurality of raisedridges 310, or raised posts/pillars on the bonding surface of thechannel bearing substrate. The upper substrate 302 is mated to the lowersubstrate under appropriate pressure and temperature conditions. Inpreferred aspects, the applied temperature is typically at or above thetransition temperature for the lower substrate, but well below thetransition temperature of the upper substrate. Under the elevatedtemperature conditions, the focused pressure upon the texturingstructures 310 melts and spreads the texture structures and bonds withthe upper substrate 302. This is illustrated in FIG. 3B, where thecollapsed or melted texture structures 310 a form the bond point betweenthe two substrate layers 302 and 304. Although preferred aspects utilizetwo substrates having different transition temperatures, this is notnecessarily required. In particular, because the microstructures permitthe focusing of pressure on those texturing structures, lower pressuresmay be used in the thermal bonding process. As noted previously,excessive applied pressures are at least partially to blame for thechannel deformations described above. Therefore, by reducing the appliedpressures, one also reduces the severity of channel deformation.

Although the surface textured methods described herein are generally inreference to thermal bonding methods, such techniques are alsoapplicable to acoustic or sonic welding or bonding techniques. Inparticular, the raised elements of the surface texturing describedherein, generally function in a manner similar to energy directors inconventional acoustic welding techniques. In use, the substrate layersare mated together and an appropriate pressure is applied. One or bothof the substrates is then acoustically vibrated, e.g., at greater thanabout 10 to 20 KHz. The vibrational friction caused at the contact pointbetween the two surfaces, e.g., on the texture elements or ridges,results in a localized heating, melting and bonding of the substratelayers. Further, as with the thermal bonding methods, once the textureelements have completely melted or compressed into their respectivesurfaces, the applied pressure is spread over the entire surface area,and melting and bonding cease. Again, this prevents substantialdistortion of the channels. Acoustic welding methods and systems havebeen described in the art, and are commercially available, e.g., fromHermann Ultrasonics, Inc.

IV. Other Polymer Selection Criteria

In addition to selecting polymeric substrates based upon theirtransition temperatures, there are also a number of other criteria onecan apply in polymer selection. For example, the microfluidic devices ofthe present invention are often used in the performance of analyticaloperations which employ optical detection systems. Such devicestypically include a detection window disposed across one of the channelsof the device and through which an optical signal can pass. As such,polymeric materials that are transparent are generally used in thefabrication of such devices. In particularly preferred aspects,fluorescent detection systems are utilized. This generally dictates thatpolymer grades be selected that have minimal levels of background orauto-fluorescence. Typically, auto-fluorescence is lower in certainpolymer types, e.g., polymethylmethacrylate, as well as in more puregrades of polymers. Table 2 illustrates a comparison of theautofluorescence of different types and grades of polymers as comparedto different types of glass.

Selection of an appropriate polymer type and grade generally dependsupon the type of detection system utilized, the wavelength of lightapplied to the system, and the like. In general, however, the backgroundfluorescence of the polymer substrate is less than 5 times that ofglass, preferably less than twice that of glass, and more preferably,approximately the same as or less than glass, for the desiredwavelength.

In addition to detection criteria, polymer substrates are alsooptionally selected for their ability to support or eliminateelectroosmotic flow. In particular, as described in U.S. Ser. No.08/843,212 filed Apr. 14, 1997 (incorporated herein by reference for allpurposes), polymeric substrates may be selected or treated to support adesired level of electroosmotic flow, depending upon the application towhich the device is going to be put. In particular, some polymericmaterials have a sufficiently high level of surface charge to allowadequate electroosmotic flow in microscale channels fabricated fromthose materials. Electroosmotic flow is generally a desirablecharacteristic where the device is utilized in applications that employbulk fluid flow within the channel networks, whereas certain otherapplications, e.g., nucleic acid separations, generally seek toeliminate such flow. Again, polymers may be selected to achieve thislatter goal.

The present invention is illustrated in greater detail with reference tothe following nonlimiting examples.

EXAMPLES Example 1 Polymer Selection

Polymers were selected based upon their clarity, low fluorescence,processability and commercial availability. Several polymer materialswere evaluated, as set forth in Table 1, below.

TABLE 1 Plexiglas Makrolon Lexan Acrylite M-30 Acrylite L-40 VS UVTDP-1-1265 OQ1020L Property (Acrylic) (Acrylic) (Acrylic) (Polycarb.)(Polycarb.) Transmittance 92 92 92 89 90 Haze (%) <1 2 2 . . . . . .Melt Flow Rate 24 28 24 75 65 (g/10 min) (all at 230° C., 3.8 kg)Refract. Index 1.49 1.49 1.49 1.582 1.58 Dielectric 19.7 19.7 . . . >1614.8–17.6 Strength (kV/mm) Vol. Resistivity . . . . . . . . . 1.0 × 10¹⁶1.0 × 10¹⁷ (Ohm/cm) Supplier CYRO Indust. Cyro Indust. Atohaas, Bayer GEPlastics North Am.Based upon the results shown in Table 1, acrylic polymers, andparticularly polymethylmethacrylate were selected as the best polymersubstrate, with polycarbonate being the next best selection. Furthertests were performed on these polymers and the results are shown inTable 2. Polymer resins were tested using injection molded test plates.

Fluorescence was measured using the following conditions: ExcitationWavelengths 450–480 nm Emission Wavelengths 510–549 nm

TABLE 2 Fluor- Thick- escent Softening Material ness Counts Point/T_(g)PMMA Acrylite M-30 1.0 mm 1,720  90° C. Plexiglas UVT 1.0 mm 1,800 87°C./91° C. Acrylite L-40 1.0 mm 1,100  82° C. Polycarbonate MakrolonDP1-1265 1.0 mm 12,300  144° C. Lexan OQ 1020L 1.0 mm 14,800  — GlassWhite Crown 2.8 mm   500 (Hoya) White Crown 3.0 mm   400 (Schott) GreenSoda Lime 2.3 mm 1,080

Example 2 Thermal Bonding of Polymer Substrates

Initial bonding experiments utilized an embossed channel plate(substrate) fabricated from Plexiglas clear 99530 (described above). Thechannels had dimensions of 100 μm wide and 32 μm deep. A L-40 PMMA coverplate was thermally bonded to the channel plate at 84° C., the softeningpoint of the L-40 polymer, and with an applied force of approximately 10kg. Cross-sectional examination of the bonded channel showed that whilethe embossed channel plate maintained its structure, the cover plate haddeformed into the channel, as shown in FIG. 5A. The provided dimensionsare approximate. The bonding temperature was then adjusted to 80° C.,and the experiment repeated. In this latter experiment, the crosssection of the bonded parts showed that the channel had achieved a goodseal, the channel was not distorted, nor had the cover platesubstantially flowed into the channel as shown in FIG. 5B.

All publications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference. Although the present invention has beendescribed in some detail by way of illustration and example for purposesof clarity and understanding, it will be apparent that certain changesand modifications may be practiced within the scope of the appendedclaims.

1. A method of fabricating a microfluidic device, comprising: providinga first substrate having at least a first surface and a second substratehaving at least a first surface, wherein at least one of the firstsurface of the first substrate or the first surface of the secondsubstrate comprises a textured surface having a plurality of texturingstructures fabricated thereon, the textured surface not comprising anapplied adhesive, and wherein at least one of the first surface of thefirst substrate or the first surface of the second substrate comprises agroove fabricated therein, the groove not forming a portion of thetexturing structures; and mating and bonding the first surface of thefirst substrate to the first surface of the second substrate, while atthe same time deforming the textured surface, the groove defining achannel when the first and second substrates are mated and bonded. 2.The method of claim 1, wherein the texturing structures comprise aplurality of raised ridges.
 3. The method of claim 1, wherein thetexturing structures comprises a plurality of microscale posts.
 4. Themethod of claim 1, wherein the step of mating and bonding comprisesapplying heat and pressure to the first and second substrates tothermally bond the first surface of the first substrate to the firstsurface of the second substrate.
 5. The method of claim 1, wherein atleast one of the first surface of the first substrate and the firstsurface of the second substrate comprises a plurality of groovesfabricated therein, the grooves defining an integrated channel networkwhen the first and second substrates are mated and bonded, and whereinthe texturing structures have a height that is no greater than 50% of adepth of the grooves.
 6. The method of claim 1, wherein at least one ofthe first surface of the first substrate and the first surface of thesecond substrate comprises a plurality of grooves fabricated therein,the grooves defining an integrated channel network when the first andsecond substrates are mated and bonded, and wherein the texturingstructures have a height that is between about 1% and about 50% of adepth of the grooves.
 7. The method of claim 1, wherein at least one ofthe first surface of the first substrate and the first surface of thesecond substrate comprises a plurality of grooves fabricated therein,the grooves defining an integrated channel network when the first andsecond substrates are mated and bonded, and wherein the texturingstructures have a height that is between about 1% and about 30% of adepth of the grooves.
 8. The method of claim 1, wherein at least one ofthe first surface of the first substrate and the first surface of thesecond substrate comprises a plurality of grooves fabricated therein,the grooves defining an integrated channel network when the first andsecond substrates are mated and bonded, and wherein the texturingstructures have a height that is between about 1% and about 10% of adepth of the grooves.
 9. The method of claim 1, wherein at least one ofthe first surface of the first substrate and the first surface of thesecond substrate comprises a plurality of grooves fabricated therein,the grooves defining an integrated channel network when the first andsecond substrates are mated and bonded, and wherein the texturingstructures have a height that is from about 0.25 μm to about 50 μm high.10. The method of claim 1, wherein at least one of the first surface ofthe first substrate and the first surface of the second substratecomprises a plurality of grooves fabricated therein, the groovesdefining an integrated channel network when the first and secondsubstrates are mated and bonded, and wherein the texturing structureshave a height that is from about 0.25 μm to about 30 μm high.
 11. Themethod of claim 1, wherein at least one of the first surface of thefirst substrate and the first surface of the second substrate comprisesa plurality of grooves fabricated therein, the grooves defining anintegrated channel network when the first and second substrates aremated and bonded, and wherein the texturing structures have a heightthat is from about 0.25 μm to about 10 μm high.
 12. The method of claim1, wherein at least one of the first surface of the first substrate andthe first surface of the second substrate comprises a plurality ofgrooves fabricated therein, the grooves defining an integrated channelnetwork when the first and second substrates are mated and bonded, andwherein the texturing structures have a height that is from about 0.5 μmto about 2 μm high.
 13. The method of claim 1, wherein the step ofmating and bonding comprises ultrasonically welding the first surface ofthe first substrate to the first surface of the second substrate. 14.The method of claim 1, wherein the step of mating and bonding comprisesthermally bonding the first surface of the first substrate to the firstsurface of the second substrate.
 15. The method of claim 1, wherein thestep of mating and bonding comprises sonically bonding the first surfaceof the first substrate to the first surface of the second substrate.